Title

Expression of a cDNA derived from the yeast killer preprotoxin gene: implications for processing and immunity

UMMS Affiliation

Department of Molecular Genetics and Microbiology; Department of Microbiology and Physiological Systems

Publication Date

1986-03-01

Document Type

Article

Disciplines

Immunity | Microbiology | Physiology

Abstract

The type I killer strains of Saccharomyces cerevisiae secrete a dimeric 19-kDa protein that kills sensitive cells by disrupting cytoplasmic membrane function. This toxin is encoded by the double-stranded RNA plasmid M1-dsRNA, which also determines specific immunity to toxin. A preprotoxin, the 35-kDA in vitro translation product of denatured M1-dsRNA, is presumed to be the primary in vivo gene product. To facilitate studies on preprotoxin structure and maturation, we have inserted a partial cDNA copy of M1-dsRNA into the yeast vector p1A1, bringing it under control of the phosphate-repressible PHO5 promoter. This in-frame gene fusion encodes all of the preprotoxin sequence except for its N-terminal secretion leader, which is replaced by the leader sequence of PHO5. Transformation of sensitive yeast strains lacking M1-dsRNA with such fusion plasmids converts them to phosphate-repressible, immune killers, demonstrating that both toxin and immunity determinants are contained within the preprotoxin molecule. L-1-Tosylamido-2-phenylethyl chloromethyl ketone retards glycosylation of preprotoxin to toxin, facilitating size comparisons and indicating that processing of the normal precursor involves three glycosylation events but does not involve cotranslational leader peptidase action. In contrast, the PHO5 leader is apparently removed from the fusion preprotoxin.

DOI of Published Version

10.1073/pnas.83.6.1675

Source

Proc Natl Acad Sci U S A. 1986 Mar;83(6):1675-9. doi: 10.1073/pnas.83.6.1675. Link to article on publisher's site

Journal/Book/Conference Title

Proceedings of the National Academy of Sciences of the United States of America

Related Resources

Link to Article in PubMed

PubMed ID

3513184

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